Article 101

Ozone Partially Prevents Diabetic Neuropathy in Rats

Authors

H. A. Erken1, O. Genç2, G. Erken1, C. Ayada2, G. Gündoğdu3, H. Doğan4

Affiliations

1

Key words ▶ diabetes ● ▶ insulin ● ▶ neuropathy ● ▶ ozone ● ▶ oxidative stress ●

Abstract

received 24.06.2014 first decision 13.08.2014 accepted 27.08.2014

Introduction

Correspondence H. A. Erken, PhD Department of Physiology Faculty of Medicine Balikesir University Balikesir Turkey Tel.:  + 90/266/6121 461 Fax:  + 90/266/6121 459 [email protected]



Neuropathy is one of the most common complications of diabetes mellitus. Although the beneficial effects of good blood glucose control on diabetic neuropathy are known, this control cannot completely prevent the occurrence and progression of diabetic neuropathy. The aim of this study was to investigate whether ozone prevents diabetic neuropathy. 36 adult female SpragueDawley rats were randomly divided into 6 groups (n = 6): control (C), ozone (O), diabetic (D), ozonetreated diabetic (DO), insulin-treated diabetic (DI), and ozone- and insulin-treated diabetic (DOI). Diabetes was induced by a single injection of streptozotocin (60  mg/kg, intraperitoneal [i.p.]), after which insulin was administered (3 IU, i.p.) to the DI and DOI groups for 28 days, and



The number of patients with diabetes is increasing rapidly, and the prevalence of diabetes in the 20–79 age groups is estimated to be 10.1 % worldwide in 2035. Diabetes mellitus is a chronic metabolic disorder that leads to long-term complications that affect tissues such as the retina, kidney, heart, and peripheral nerves [1]. Peripheral neuropathy occurs in more than 50 % of diabetic patients. Early disorders of nerve function include slowing of the nerve conduction velocity (NCV), followed by axonal degeneration, paranodal demyelination, and loss of myelinated fibres. Small nerve fibre neuropathies occur during the early stages of diabetes and frequently develop with no objective signs or electrophysiological evidence of nerve damage. As the disease progresses, the nerve degeneration symptoms present as a slowing of NCV, a decrease in the amplitude of compound action potential (CAP), a reduction in thermal sensitivity, and altered activity of Na + /K + -ATPase, which is

1.1 mg/kg (50 µg/ml) ozone was given to the O, DO, and DOI groups for 15 days. 4 weeks after the induction of diabetes, the nerve conduction velocity (NCV), amplitude of the compound action potential (CAP), total oxidant status (TOS), and total antioxidant status (TAS) were measured, and the oxidative stress index (OSI) was calculated. The NCV, amplitude of CAP, and TAS of the DI and DOI groups were higher than those of the D group; the amplitudes of CAP and TAS of the DO group were higher than those of the D group; and the TOS and OSI of the DO, DI, and DOI groups were lower than those of the D group. These findings indicate that ozone partially prevents diabetic neuropathy in rats. It appears that the preventive effects of ozone are mediated through oxidant/antioxidant mechanisms.

responsible for the maintenance of the potential difference throughout nerves [2, 3]. Ozone has a molecular weight of 48, and its solubility in water is approximately 10 times higher than that of oxygen. Although ozone dissolves rapidly in pure water and obeys Henry’s law in biological water, ozone instantly reacts with the inorganic and organic molecules dissolved in the water, generating a variety of free radicals [4]. However, it can exert protective effects through oxidative preconditioning, stimulating and/or preserving the endogenous antioxidant systems, and blocking the xanthine/xanthine oxidase pathway for reactive oxygen species generation [5, 6]. It has been shown that ozone therapy has a beneficial effect on blood lipid metabolism by reducing the concentration of blood cholesterol and activating the antioxidant protection system [7]. Due to its germicidal properties and its influence on the processes of oxygen metabolism, as well as its other effects, ozone has been used with good results to treat patients with diabetic foot [8].

Erken HA et al. Ozone Prevents Diabetic Neuropathy …  Exp Clin Endocrinol Diabetes 2015; 123: 101–105

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1389954 Published online: December 11, 2014 Exp Clin Endocrinol Diabetes 2015; 123: 101–105 © J. A. Barth Verlag in Georg Thieme Verlag KG ­Stuttgart · New York ISSN 0947-7349

 Department of Physiology, Faculty of Medicine, Balikesir University, Balikesir, Turkey  Department of Physiology, Faculty of Medicine, Dumlupinar University, Kutahya, Turkey 3  Denizli State Hospital, Denizli, Turkey 4  Ozone Treatment Center, Denizli, Turkey 2

102 Article

Materials and Methods



All experimental protocols conducted on the animals were consistent with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85-23) and approved by the local Ethics Committee on Animal Care. 36 adult female Sprague-Dawley rats weighing 259 ± 14 g (mean ±  SD) were used. All of the rats were maintained in a 12-h light/ dark cycle environment (lights on 7:00–19:00 h) at 22 ± 1 °C and 50 % humidity, and they were kept in transparent plastic cages (42 × 26 × 15 cm), which each contained 3 rats. The rats had access to food and water ad libitum. The rats were randomly divided into 6 groups (n = 6): control (C), ozone (O), diabetic (D), ozone-treated diabetic (DO), insulintreated diabetic (DI), and ozone- and insulin-treated diabetic (DOI). Diabetes was induced by a single intraperitoneal (i.p.) injection of freshly prepared STZ (Sigma-Aldrich Co., Taufkirchen, Germany) solution (60 mg/kg body weight in 0.09 M citrate buffer, pH 4.8) in the D, DO, DI, and DOI groups [18]. The animals in the C and O groups received the same volume of vehicle. Hyperglycaemia was confirmed 48 h after STZ injection by measuring the tail vein blood glucose levels using a glucometer (Accu-Chek; Roche Diagnostics Co., Mannheim, Germany). Only the animals with mean plasma glucose levels above 300 mg/dl were classified as diabetic. The weights and blood glucose levels of all of the rats were measured before the experimental procedure and at the end of the experiments. The ozone was generated by an ozone generator (Dr. J. Hänsler OZONOSAN GmbH, Iffezheim, Germany). A 50 μg/ml concentration of ozone was given to the O, DO, and DOI groups (1.1 mg/kg i.p., 1-min injection period once a day for 15 days). The schedule and dosage of ozone used in this study has proved to be optimal in a previous study [5]. This dose of ozone has been shown to achieve oxidative preconditioning without appreciable toxicity [5, 20]. Insulin (3 IU) (Novo Nordisk Co., Bagsvaerd, Denmark) was administered to the DI and DOI groups (i.p. in 1 ml saline, once a day for

28 days). Oxygen was injected as the vehicle for ozone in the C, D, and DI groups every day for 15 days. In addition, 1 ml saline was injected in the C, O, D, and DO groups every day for 28 days.

Electrophysiological recordings

At the end of the 4-week experimental period, the rats were anaesthetised with ketamine and xylazine (90 and 10 mg/kg, respectively, i.p.), and the right hind limbs of rats were shaved and disinfected with 10 % povidone-iodine (Batticon, Adeka Co., Samsun, Turkey). The right sciatic nerves were removed and placed in Ringer’s solution. Later, the nerve fibres were placed in a nerve chamber, and the proximal end of the sciatic nerve was stimulated electrically (10 V, 0.15 ms) 30 times at 1-s intervals with a PowerLab stimulator (ADInstruments Co., Sydney, Australia). Evoked CAPs were recorded using a PowerLab 8 S/P data acquisition system and the Scope program (ADInstruments Co.). All experiments were conducted at room temperature (controlled at 24 ± 1 °C). The amplitudes of the CAPs were measured from the baseline to the peak, and the latencies were measured from the stimulus artefact to the beginning of the first deflection from the baseline. NCV values were calculated according to the following formula: NCV (m/s) = distance between stimulating and recording point – 15 mm (m)/latency (s). The average NCV value and CAP amplitude taken from each rat were considered the defined NCV and CAP amplitude values of that rat.

Measurement of plasma total oxidant status

The abdomens of the rats were opened using a midline incision while the rats were under anaesthesia, and blood from the abdominal aorta was collected in citrated tubes. The animals were then euthanised by exsanguination while under ketamine and xylazine anaesthesia. The blood samples taken from all of the rats were centrifuged at 1 500 × g for 10 min, and the plasma was separated and stored at  − 80 °C until analysis. Plasma total oxidant status (TOS) was measured using a novel automated colorimetric measurement method for TOS developed by Erel [21]. In this method, the oxidants present in the sample oxidise the ferrous ion–o–dianisidine complex to a ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundant in the reaction medium. The ferric ion produces a coloured complex with xylenol orange in an acidic medium. The colour intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules (lipids and proteins) in the sample. The assay is calibrated with hydrogen peroxide, and the results are expressed in terms of micromolar hydrogen peroxide equivalent per litre (μmol H2O2 equiv/L).

Measurement of plasma total antioxidant status

Plasma total antioxidant status (TAS) was measured using a novel automated colorimetric measurement method for TAS developed by Erel [22]. In this method, the hydroxyl radical, the most potent biological radical, is produced by a Fenton reaction and reacts with the colourless substrate o-dianisidine to produce the dianisyl radical, which is bright yellowish-brown. Upon the addition of a plasma sample, the oxidative reactions initiated by the hydroxyl radicals in the reaction mix are suppressed by the antioxidant components of the plasma, preventing the colour change and thereby providing an effective measure of the TAS of the plasma. The assay results are expressed as mmol Trolox equiv/L.

Erken HA et al. Ozone Prevents Diabetic Neuropathy …  Exp Clin Endocrinol Diabetes 2015; 123: 101–105

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

The streptozotocin (STZ)-induced diabetes model is one of the most widely used experimental models of human diabetic neuropathy. The functional, pathological, and biochemical changes that occur in this model are similar to the findings in human patients with diabetes [9]. In particular, gradual decreases in NCV and Na + /K + -ATPase activity have been observed in the sciatic nerves of STZ-treated rats [10, 11]. A therapeutic strategy that can counteract diabetic neuropathy is not currently available, and the clinical treatments are limited to good blood glucose control [12]. Promising treatments, such as metabolic treatments [12], autoimmune therapies, nerve growth factors [13], neuroactive steroids [14], acetyl-L-carnitine [15], γ-linolenic acid, docosahexaenoic acid [16], α-lipoic acid [17], progesterone, testosterone, reproductive steroid derivatives, and dehydroepiandrosterone [11, 18, 19], have been suggested for diabetic peripheral neuropathy. However, the occurrence and progression of diabetic neuropathy cannot be completely prevented by the current treatment approaches. To the best of our knowledge, no clinical or experimental studies regarding the relationship between ozone and diabetic neuropathy have been conducted. The aim of this study was to investigate the effect of ozone on STZ-induced diabetic neuropathy in rats.

Article 103 Calculation of oxidative stress index

cantly higher than that of the D group, the increase in the NCV of the DO group reached borderline significance (p = 0.058) compared with that of the D group. The NCV and CAP amplitude values of the DI and DOI groups were significantly higher than those of the D group, and the CAP amplitude of the DO group was significantly lower than those of the C and O groups. There were no significant differences in the NCV or CAP amplitude val▶  Fig. 1a, b). ues among the DO, DI, and DOI groups ( ●

The ratio of TOS to TAS is referred to as the oxidative stress index (OSI). The OSI is calculated according to the following formula: OSI (arbitrary unit) = TOS (μmol H2O2 equiv/L)/TAS (mmol Trolox equiv/L).

Statistical analysis

One-way ANOVA and Tukey’s post hoc test were used for comparisons of the experimental groups. All results are expressed as the mean ± SD, and p 

Ozone partially prevents diabetic neuropathy in rats.

Neuropathy is one of the most common complications of diabetes mellitus. Although the beneficial effects of good blood glucose control on diabetic neu...
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